Extracellular nucleotides interacting with specific nucleotide receptors (P2 receptors) participate in a wide variety of important physiological responses. One of the first recognized physiological roles of extracellular nucleotides was the aggregation of platelets induced by ADP. Adenine nucleotide effects of platelet function result from the activation of at least three different receptor subtypes: P2X1, P2Y1 and P2Y AC receptors. In spite of significant advances in the characterization of the receptors involved, the effects of nucleotides and associated signaling mechanisms remain poorly understood. ADP-induced platelet aggregation requires the simultaneous activation of P2Y1 and P2Y AC receptors, indicating that both receptor proteins are potential targets for antithrombotic agents. The affects of adenine nucleotides on platelet receptors are strongly influenced by the complex metabolism of extracellular nucleotides by ubiquitous ecto- nucleotidases. Therefore, an integrated study of receptor signaling and nucleotide metabolism is necessary to understand the regulation of platelet function by extracellular nucleotides. We have studied extensively in C5 rat glioma cells a P2Y receptor coupled to inhibition of adenylyl cyclase, and confirmed that the pharmacological and signaling properties of this receptor are identical to those of the platelet P2Y AC receptor. In spite of great efforts, the molecular identification of both, the platelet and C6 cell P2Y AC receptors remain elusive. The aim of this research is to clone the platelet ADP receptor coupled to inhibition of adenylyl cyclase, and to establish the physiological role of the extracellular nucleotide metabolism on P2Y receptor signaling. A research plan for the cloning of the platelet P2Y AC receptor involving expression-cloning strategies using the c6 P2Y AC receptor as model system are outlined. The study of extracellular nucleotide metabolism on the signaling of platelet and other P2Y receptors will follow from the progress of the previous funding period. We are now in the position to ask molecular questions about the functional interaction of P2Y receptors and extracellular nucleotide metabolism. Cloning the P2Y AC receptor and understanding the regulation of nucleotide metabolism are essential steps towards the understanding of the complex molecular mechanism of ADP-mediated platelet activation. This information should prove useful in the development of new biochemical and pharmacological tools with potential use in the treatment of coagulation-related diseases.